Polypeptides having glucoamylase activity and polynucleotides encoding same

ABSTRACT

The present invention provides isolated polypeptides having glucoamylase activity, catalytic domains, and polynucleotides encoding the polypeptides, catalytic domains. The invention also provides nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/CN2012/081124 filed Sep. 7, 2012, which claims priority or thebenefit under 35 U.S.C. 119 of International application no.PCT/CN11/079528 filed Sep. 9, 2011 and U.S. provisional application No.61/538,995 filed Sep. 26, 2011, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides having glucoamylaseactivity and polynucleotides encoding the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the polynucleotides as well as methods for producing andusing the polypeptides, and to the use of glucoamylases of the inventionfor starch conversion to producing fermentation products, such asethanol, and syrups, such as glucose. The invention also relates to acomposition comprising a glucoamylase of the invention.

Description of the Related Art

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeast, withthose from Aspergillus being commercially most important.

Commercially, glucoamylases are used to convert starchy material, whichis already partially hydrolyzed by an alpha-amylase, to glucose. Theglucose may then be converted directly or indirectly into a fermentationproduct using a fermenting organism. Examples of commercial fermentationproducts include alcohols (e.g., ethanol, methanol, butanol,1,3-propanediol); organic acids (e.g., citric acid, acetic acid,itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid,succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂), and morecomplex compounds, including, for example, antibiotics (e.g., penicillinand tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂,beta-carotene); hormones, and other compounds which are difficult toproduce synthetically. Fermentation processes are also commonly used inthe consumable alcohol (e.g., beer and wine), dairy (e.g., in theproduction of yogurt and cheese) industries.

The end product may also be syrup. For instance, the end product may beglucose, but may also be converted, e.g., by glucose isomerase tofructose or a mixture composed almost equally of glucose and fructose.This mixture, or a mixture further enriched with fructose, is the mostcommonly used high fructose corn syrup (HFCS) commercialized throughoutthe world. Amlacher et al., 2011 (Cell 146, 277-289) reports the genomeof Chaetomium thermophilum. It is an object of the present invention toprovide polypeptides having glucoamylase activity and polynucleotidesencoding the polypeptides and which provide a high yield in fermentationproduct production processes, such as ethanol production processes,including one-step ethanol fermentation processes from un-gelatinizedraw (or uncooked) starch.

SUMMARY OF THE INVENTION

Polypeptides produced by the fungus Chaetomium and having glucoamylaseactivity have been identified and characterized. More particularly theChaetomium sp. is selected from Chaetomium thermophilum.

The present invention relates to isolated polypeptides havingglucoamylase activity, selected from the group consisting of:

(a) a polypeptide having at least 76% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 76%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3, or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4comprising a substitution, deletion, and/or insertion at one or morepositions; and (e) a fragment of the polypeptides of (a), (b), (c), or(d) that has glucoamylase activity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 76% sequence identity to aminoacids 33 to 505 of SEQ ID NO: 2 or amino acids 28 to 504 of SEQ ID NO:4;

(b) a catalytic domain encoded by a polynucleotide that hybridizes undermedium stringency conditions with (i) nucleotides 97 to 1609 of SEQ IDNO: 1 or nucleotides 82 to 1680 of SEQ ID NO: 3, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a catalytic domain encoded by a polynucleotide having at least 76%sequence identity to nucleotides 97 to 1609 of SEQ ID NO: 1 ornucleotides 82 to 1680 of SEQ ID NO: 3 or the cDNA sequences thereof;

(d) a variant of amino acids 33 to 505 of SEQ ID NO: 2 or amino acids 28to 504 of SEQ ID NO: 4 comprising a substitution, deletion, and/orinsertion at one or more (e.g., several) positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that hasglucoamylase activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods of producing afermentation product from starch containing material and to compositionscomprising the glucoamylase of the invention.

DEFINITIONS

Glucoamylase: The term glucoamylase (1,4-alpha-D-glucan glucohydrolase,EC 3.2.1.3) is defined as an enzyme, which catalyzes the release ofD-glucose from the non-reducing ends of starch or related oligo- andpolysaccharide molecules. For purposes of the present invention,glucoamylase activity is determined according to the procedure describedin the ‘Materials & Methods’-section herein.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, preferably at least 45%, more preferably at least 50%,more preferably at least 55%, more preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 100% of the glucoamylase activity ofthe mature polypeptide of SEQ ID NO: 2 or at least 20%, preferably atleast 40%, preferably at least 45%, more preferably at least 50%, morepreferably at least 55%, more preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the glucoamylase activity of the maturepolypeptide of SEQ ID NO: 4.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Binding domain: The term “carbohydrate binding domain” means the regionof an enzyme that mediates binding of the enzyme to the carbohydratesubstrate. The carbohydrate binding domain (CBD) is typically foundeither at the N-terminal or at the C-terminal extremity of anglucoamylase. The CBD is also sometimes referred to as a starch bindingdomain, SBD. In one embodiment the CBD is amino acids 526 to 634 of SEQID NO: 2 or amino acids 510 to 631 of SEQ ID NO: 4.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme. In oneembodiment the catalytic domain is amino acids 33 to 505 of SEQ ID NO: 2or amino acids 28 to 504 of SEQ ID NO: 4.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide or a catalytic domainhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or domain; wherein thefragment has glucoamylase activity. In one aspect, a fragment containsat least 473-amino acid residues (e.g., amino acids 33 to 505 of SEQ IDNO: 2), or at least 477-amino acid residues (e.g., amino acids 28 to 504of SEQ ID NO: 4).

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 20 to 634 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6) thatpredicts amino acids 1 to 19 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 20 to 631 of SEQID NO: 4 based on the SignalP program (Nielsen et al., 1997, ProteinEngineering 10:1-6) that predicts amino acids 1 to 19 of SEQ ID NO: 4are a signal peptide.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving glucoamylase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1999 (including the stop codon) ofSEQ ID NO: 1 or the cDNA sequence thereof based on the SignalP (Nielsenet al., 1997, supra) that predicts nucleotides 1 to 57 of SEQ ID NO: 1encode a signal peptide. In another aspect the mature polypeptide codingsequence is nucleotides 58-241, and 336-1996 of SEQ ID NO 1.

In another aspect, the mature polypeptide coding sequence is nucleotides58 to 2064 (including the stop codon) of SEQ ID NO: 3 or the cDNAsequence thereof based on the SignalP (Nielsen et al., 1997, supra) thatpredicts nucleotides 1 to 57 of SEQ ID NO: 3 encode a signal peptide. Inanother aspect the mature polypeptide coding sequence is nucleotides58-103, 160-273, 332-701, and 756-2061 of SEQ ID NO 3.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes of the present invention, the sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM 62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment—Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment—Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having glucoamylase activity. In one aspect, a subsequencecontains at least 1419 nucleic acid residues (e.g., nucleotides 97 to241, 336 to 1609 of SEQ ID NO: 1), or at least 1431 nucleic acidresidues (e.g., nucleotides 82 to 103, 160 to 273, 332 to 701, 756 to1680 of SEQ ID NO: 3).

Variant: The term “variant” means a polypeptide having glucoamylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Glucoamylase Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 76%, at least 78%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveglucoamylase activity. In one aspect, the polypeptides differ by no morethan 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the maturepolypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having glucoamylase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the polypeptidecomprises or consists of amino acids 20 to 634 of SEQ ID NO: 2.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 76%, at least 78%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveglucoamylase activity. In one aspect, the polypeptides differ by no morethan 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the maturepolypeptide of SEQ ID NO: 4.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 4 or an allelic variantthereof; or is a fragment thereof having glucoamylase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 4. In another aspect, the polypeptidecomprises or consists of amino acids 20 to 631 of SEQ ID NO: 4.

In another embodiment, the present invention relates to an isolatedpolypeptide having glucoamylase activity encoded by a polynucleotidethat hybridizes under medium, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

In another embodiment, the present invention relates to an isolatedpolypeptide having glucoamylase activity encoded by a polynucleotidethat hybridizes under medium, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 3, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3, or a subsequencethereof, as well as the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, ora fragment thereof, may be used to design nucleic acid probes toidentify and clone DNA encoding polypeptides having glucoamylaseactivity from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having glucoamylase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequencethereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1 or SEQ ID NO: 3; (ii) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3; (iii) the cDNA sequencesthereof; (iv) the full-length complement thereof; or (v) a subsequencethereof; under medium to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is nucleotides 58-241, and336-1996 of SEQ ID NO 1. In another aspect, the nucleic acid probe is apolynucleotide that encodes the polypeptide of SEQ ID NO: 2; the maturepolypeptide thereof; or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 1 or the cDNA sequence thereof.

In one aspect, the nucleic acid probe is nucleotides 58-103, 160-273,332-701, and 756-2061 of SEQ ID NO 3. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 4; the mature polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 3 or the cDNA sequencethereof.

In another embodiment, the present invention relates to an isolatedpolypeptide having glucoamylase activity encoded by a polynucleotidehaving a sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1 or the cDNA sequence thereof of at least 76%, at least 78%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to an isolatedpolypeptide having glucoamylase activity encoded by a polynucleotidehaving a sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 3 or the cDNA sequence thereof of at least 76%, at least 78%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In an embodiment, the number of amino acid substitutions,deletions and/or insertions introduced into the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4 is not more than 10, e.g., 1, 2, 3, 4, 5,6, 7, 8 or 9. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for glucoamylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al, 2000, J.Biotechnol. 76: 245-251; Rasmussen-Wilson et al, 1997, Appl. Environ.Microbiol. 63: 3488-3493; Ward et al, 1995, Biotechnology 13: 498-503;and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al.,1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Glucoamylase Activity

A polypeptide having glucoamylase activity of the present invention maybe obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a Chaetomium polypeptide.

In another aspect, the polypeptide is a Chaetomium thermophilumpolypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 33 to 505 of SEQ IDNO: 2 of at least 76%, at least 78%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In one aspect, the catalytic domains comprise aminoacid sequences that differ by no more than 10 amino acids, e.g., 1, 2,3, 4, 5, 6, 7, 8, or 9, from amino acids 33 to 505 of SEQ ID NO: 2.

The catalytic domain preferably comprises or consists of amino acids 33to 505 of SEQ ID NO: 2 or an allelic variant thereof; or is a fragmentthereof having glucoamylase activity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides that hybridize under mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions (as definedabove) with (i) the nucleotides 97 to 1624 of SEQ ID NO: 1, (ii) thecDNA sequence thereof, or (iii) the full-length complement of (i) or(ii) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 97 to 1609 of SEQ ID NO: 1 or the cDNA sequence thereof ofat least 76%, at least 78%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 97 to 1609 of SEQ ID NO: 1 In anotherembodiment, the present invention also relates to catalytic domainvariants of amino acids 33 to 505 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids33 to 505 of SEQ ID NO: 2 is 10, e.g., 1, 2, 3, 4, 5, 6, 8, or 9.

In another embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 28 to 504 of SEQ IDNO: 4 of at least 76%, at least 78%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In one aspect, the catalytic domains comprise aminoacid sequences that differ by no more than 10 amino acids, e.g., 1, 2,3, 4, 5, 6, 7, 8, or 9, from amino acids 28 to 504 of SEQ ID NO: 4.

The catalytic domain preferably comprises or consists of amino acids 28to 504 of SEQ ID NO: 4 or an allelic variant thereof; or is a fragmentthereof having glucoamylase activity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides that hybridize under mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions (as definedabove) with (i) the nucleotides 82 to 1680 of SEQ ID NO: 3, (ii) thecDNA sequence thereof, or (iii) the full-length complement of (i) or(ii) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 82 to 1680 of SEQ ID NO: 3 or the cDNA sequence thereof ofat least 76%, at least 78%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 82 to 1680 of SEQ ID NO: 3 In anotherembodiment, the present invention also relates to catalytic domainvariants of amino acids 28 to 504 of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids31 to 430 of SEQ ID NO: 4 is 10, e.g., 1, 2, 3, 4, 5, 6, 8, or 9.

The catalytic domain according to the invention may in one embodimentfurther comprise a carbohydrate binding domain.

Binding Domains

In one embodiment, the present invention also relates to carbohydratebinding domains having a sequence identity to amino acids 526 to 634 ofSEQ ID NO: 2 of at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In one aspect, the carbohydrate binding domains comprise aminoacid sequences that differ by no more than 10 amino acids, e.g., 1, 2,3, 4, 5, 6, 7, 8, or 9, from amino acids 526 to 634 of SEQ ID NO: 2.

The carbohydrate binding domain preferably comprises or consists ofamino acids 526 to 634 of SEQ ID NO: 2 or an allelic variant thereof; oris a fragment thereof having carbohydrate binding activity.

In another embodiment, the present invention also relates tocarbohydrate binding domains encoded by polynucleotides that hybridizeunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions (as definedabove) with (i) the nucleotides 1670 to 1996 of SEQ ID NO: 1, or (ii)the full-length complement of (i) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates tocarbohydrate binding domains encoded by polynucleotides having asequence identity to nucleotides 1670 to 1996 of SEQ ID NO: 1 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%.

The polynucleotide encoding the carbohydrate binding domain preferablycomprises or consists of nucleotides 1670 to 1996 of SEQ ID NO: 1.

In another embodiment, the present invention also relates tocarbohydrate binding domain variants of amino acids 526 to 634 of SEQ IDNO: 2 comprising a substitution, deletion, and/or insertion at one ormore (e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 526 to 634 of SEQ ID NO: 2 is 10, e.g., 1, 2, 3, 4, 5, 6,8, or 9.

In another embodiment, the present invention also relates tocarbohydrate binding domains having a sequence identity to amino acids510 to 631 of SEQ ID NO: 4 of at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In one aspect, the carbohydrate binding domainscomprise amino acid sequences that differ by no more than 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from amino acids 510 to 631of SEQ ID NO: 4.

The carbohydrate binding domain preferably comprises or consists ofamino acids 510 to 631 of SEQ ID NO: 4 or an allelic variant thereof; oris a fragment thereof having carbohydrate binding activity.

In another embodiment, the present invention also relates tocarbohydrate binding domains encoded by polynucleotides that hybridizeunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions (as definedabove) with (i) the nucleotides 1696 to 2061 of SEQ ID NO: 3, or (ii)the full-length complement of (i) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates tocarbohydrate binding domains encoded by polynucleotides having asequence identity to nucleotides 1696 to 2061 of SEQ ID NO: 3 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%.

The polynucleotide encoding the carbohydrate binding domain preferablycomprises or consists of nucleotides 1696 to 2061 of SEQ ID NO: 3.

In another embodiment, the present invention also relates tocarbohydrate binding domain variants of amino acids 510 to 631 of SEQ IDNO: 4 comprising a substitution, deletion, and/or insertion at one ormore (e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 510 to 631 of SEQ ID NO: 4 is 10, e.g., 1, 2, 3, 4, 5, 6,8, or 9.

A catalytic domain operably linked to the carbohydrate binding domain ofthe invention may be from a hydrolase, isomerase, ligase, lyase,oxidoreductase, or transferase, e.g., an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolyticenzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, xylanase, or beta-xylosidase. In oneparticular embodiment the catalytic domain is from a glucoamylase. Inanother particular embodiment the catalytic domain is from an amylase.The polynucleotide encoding the catalytic domain may be obtained fromany prokaryotic, eukaryotic, or other source.

Hybrid Enzymes

The present invention also relates to hybrid enzymes comprising acatalytic domain having enzyme activity (e.g., starch degrading enzymeactivity, such as alpha-amylase, amylopullulanase, beta-amylase, CGTase,glucoamylase, isoamylase, maltogenic amylase, or pullulanase activity),and a carbohydrate-binding domain (CBD). The hybrid enzyme may furthercomprise a linker. In one embodiment the catalytic domain is a catalyticdomain according to the invention. In another embodiment the CBD is aCBD according to the invention.

The hybrid may be produced by fusing a first DNA sequence encoding acatalytic domain and a second DNA sequence encoding acarbohydrate-binding module, or the hybrid may be produced as acompletely synthetic gene based on knowledge of the amino acid sequencesof suitable CBDs, linkers and catalytic domains.

The term “hybrid enzyme” (also referred to as “fusion protein”,“hybrid”, hybrid polypeptide” or “hybrid protein”) is used herein tocharacterize the hybrid polypeptides of the invention comprising acatalytic module having enzyme activity (e.g., starch degrading enzymeactivity, such as alpha-amylase, amylopullulanase, beta-amylase, CGTase,glucoamylase, isoamylase, maltogenic amylase, or pullulanase activity)and a carbohydrate-binding module wherein the catalytic domain and thecarbohydrate-binding module are derived from different sources. The term“source” includes, but is not limited to, a parent enzyme or a variantthereof, e.g., an amylase or glucoamylase, or other catalytic activitycomprising a suitable catalytic module and/or a suitable CBD and/or asuitable linker. However the CBD may also be derived from a polypeptidehaving no catalytic activity. The catalytic domain and the carbohydratebinding module may be derived from the same microbial strain, fromstrains within the same species, from closely related species or lessrelated organisms. Preferably the catalytic domain and the carbohydratebinding module of the hybrids are derived from different sources, e.g.,from different enzymes from the same strain and/or species, or e.g.,from strains within different species.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide, a catalytic domain, or carbohydrate binding domain of thepresent invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al, 1990,PCR: A Guide to Methods and Application, Academic Press, New York. Othernucleic acid amplification procedures such as ligase chain reaction(LCR), ligation activated transcription (LAT) and polynucleotide-basedamplification (NASBA) may be used. The polynucleotides may be clonedfrom a strain of Chaetomium, or a related organism and thus, forexample, may be an allelic or species variant of the polypeptideencoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:3, or the cDNA sequences thereof, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions that do not result in a changein the amino acid sequence of the polypeptide, but which correspond tothe codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions that may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al, 1991, Protein Expressionand Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reeseixylanase I, Trichodermareeseixylanase II, Trichoderma reesei beta-xylosidase, as well as theNA2-tpi promoter (a modified promoter from an Aspergillus neutralalpha-amylase gene in which the untranslated leader has been replaced byan untranslated leader from an Aspergillus triose phosphate isomerasegene; non-limiting examples include modified promoters from anAspergillus niger neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillusnidulans or Aspergillus oryzae triose phosphate isomerase gene); andmutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al, 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rmB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of agene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al, 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus otyzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al, 1991, Gene 98: 61-67; Cullen et al, 1987,Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1gene and construction of plasmids or vectors comprising the gene can beaccomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al, 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al, 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycotaas wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyceslactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowialipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinuscinereus, Coriolushirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete Chrysosporium, Phlebiaradiata, Pleurotusetyngii, Thielaviaterrestris, Trametesvillosa, Trametesversicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al, 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bactetiol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is a Chaetomium cell. In a morepreferred aspect, the cell is a Chaetomium thermophilum cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al, 1993, supra, disclose the use of the first intron ofthe rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al, 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al, 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theglucoamylase activity of the composition has been increased, e.g., withan enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseurn, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Combination of Glucoamylase and Acid Alpha-Amylase

According to this aspect of the invention a glucoamylase of theinvention may be combined with an alpha-amylase, preferably acidalpha-amylase in a ratio of between 0.3 and 5.0 AFAU/AGU. Morepreferably the ratio between acid alpha-amylase activity andglucoamylase activity is at least 0.35, at least 0.40, at least 0.50, atleast 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, atleast 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.85, or even at least1.9 AFAU/AGU. However, the ratio between acid alpha-amylase activity andglucoamylase activity should preferably be less than 4.5, less than 4.0,less than 3.5, less than 3.0, less than 2.5, or even less than 2.25AFAU/AGU. In AUU/AGI the activities of acid alpha-amylase andglucoamylase are preferably present in a ratio of between 0.4 and 6.5AUU/AGI. More preferably the ratio between acid alpha-amylase activityand glucoamylase activity is at least 0.45, at least 0.50, at least0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least2.1, at least 2.2, at least 2.3, at least 2.4, or even at least 2.5AUU/AGI. However, the ratio between acid alpha-amylase activity andglucoamylase activity is preferably less than 6.0, less than 5.5, lessthan 4.5, less than 4.0, less than 3.5, or even less than 3.0 AUU/AGI.

Above composition is suitable for use in a starch conversion processmentioned below for producing syrup and fermentation products, such asethanol.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to processes/methods for usingthe polypeptides having glucoamylase activity of the invention.

Uses according to the invention include starch conversion of starch to,e.g., syrup and fermentation products, including ethanol and beverages.Examples of processes where a glucoamylase of the invention may be usedinclude the ones described in WO 92/20777, WO 03/066816, WO 03/066826,WO 2004/080923, and WO 2004/081193, which are hereby all incorporated byreference.

Production of Fermentation Products

Process for Producing Fermentation Products from Gelatinized StarchContaining Material

In this aspect the present invention relates to a process for producinga fermentation product, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.

The invention relates to a process for producing a fermentation productfrom starch-containing material comprising the steps of:

(a) liquefying starch-containing material; preferably using analpha-amylase

(b) saccharifying the liquefied material obtained in step (a) using aglucoamylase of the invention; and

(c) fermenting the saccharified material using a fermenting organism.

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. Suitablestarch-containing starting materials are listed in the section“Starch-containing materials”-section below. Contemplated enzymes arelisted in the “Enzymes”-section below. The liquefaction is preferablycarried out in the presence of an alpha-amylase. The fermentation ispreferably carried out in the presence of yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”—section below. In preferred embodiments step (b)and (c) are carried out sequentially or simultaneously (i.e., as SSFprocess).

In a particular embodiment, the process of the invention furthercomprises, prior to the step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling; and

y) forming a slurry comprising the starch-containing material and water.

The aqueous slurry may contain from 10-40 wt. %, preferably 25-35 wt. %starch-containing material. The slurry is heated to above thegelatinization temperature and alpha-amylase, preferably bacterialand/or acid fungal alpha-amylase, may be added to initiate liquefaction(thinning). The slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to an alpha-amylase in step(a) of the invention.

More specifically liquefaction may be carried out as a three-step hotslurry process. The slurry is heated to between 60-95° C., preferably80-85° C., and alpha-amylase is added to initiate liquefaction(thinning). Then the slurry may be jet-cooked at a temperature between95-140° C., preferably 105-125° C., for 1-15 minutes, preferably for3-10 minute, especially around 5 minutes. The slurry is cooled to 60-95°C. and more alpha-amylase is added to finalize hydrolysis (secondaryliquefaction). The liquefaction process is usually carried out at pH4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefiedwhole grains are known as mash.

The saccharification in step (b) may be carried out using conditionswell known in the art. For instance, a full saccharification process maylast up to from about 24 to about 72 hours, however, it is common onlyto do a pre-saccharification of typically 40-90 minutes at a temperaturebetween 30-65° C., typically about 60° C., followed by completesaccharification during fermentation in a simultaneous saccharificationand fermentation process (SSF process). Saccharification is typicallycarried out at temperatures from 30-65° C., typically around 60° C., andat a pH between 4 and 5, normally at about pH 4.5.

The most widely used process in fermentation product, especiallyethanol, production is the simultaneous saccharification andfermentation (SSF) process, in which there is no holding stage for thesaccharification, meaning that fermenting organism, such as yeast, andenzyme(s) may be added together. SSF may typically be carried out at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In accordance with the present invention the fermentation step (c)includes, without limitation, fermentation processes used to producealcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citricacid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones(e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ andCO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B12, beta-carotene); and hormones. Preferredfermentation processes include alcohol fermentation processes, as arewell known in the art. Preferred fermentation processes are anaerobicfermentation processes, as are well known in the art.

Processes for Producing Fermentation Products from Un-GelatinizedStarch-Containing

In this aspect the invention relates to processes for producing afermentation product from starch-containing material withoutgelatinization of the starch-containing material (i.e., uncookedstarch-containing material). In one embodiment only a glucoamylase ofthe invention is used during saccharification and fermentation.According to the invention the desired fermentation product, such asethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material. In one embodiment a processof the invention includes saccharifying (milled) starch-containingmaterial, e.g., granular starch, below the gelatinization temperature inthe presence of a glucoamylase of the invention to produce sugars thatcan be fermented into the desired fermentation product by a suitablefermenting organism.

Accordingly, in this aspect the invention relates to a process forproducing a fermentation product from starch-containing materialcomprising:

(a) saccharifying starch-containing material with a mature glucoamylaseaccording to the invention, preferably having the sequence shown asamino acids 20 to 634 of SEQ ID NO: 2, or amino acids 20 to 631 of SEQID NO: 4 or a glucoamylase having at least 90% identity thereto, at atemperature below the initial gelatinization temperature of saidstarch-containing material,

(b) fermenting using a fermenting organism.

Steps (a) and (b) of the process of the invention may be carried outsequentially or simultaneously. In an embodiment a slurry comprisingwater and starch-containing material is prepared before step (a).

The fermentation process may be carried out for a period of 1 to 250hours, preferably is from 25 to 190 hours, more preferably from 30 to180 hours, more preferably from 40 to 170 hours, even more preferablyfrom 50 to 160 hours, yet more preferably from 60 to 150 hours, even yetmore preferably from 70 to 140 hours, and most preferably from 80 to 130hours.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. Starchheated in water begins to gelatinize between 50° C. and 75° C.; theexact temperature of gelatinization depends on the specific starch, andcan readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material is the temperature atwhich birefringence is lost in 5% of the starch granules using themethod described by Gorinstein and Lii, 1992, Starch/Stärke 44(12):461-466 (1992).

Before step (a) a slurry of starch-containing material, such as granularstarch, having 10-55 wt. % dry solids, preferably 25-40 wt. % drysolids, more preferably 30-35 wt. % dry solids of starch-containingmaterial may be prepared. The slurry may include water and/or processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side stripper water from distillation, orother fermentation product plant process water. Because the process ofthe invention is carried out below the gelatinization temperature andthus no significant viscosity increase takes place, high levels ofstillage may be used if desired. In an embodiment the aqueous slurrycontains from about 1 to about 70 vol. % stillage, preferably 15-60%vol. % stillage, especially from about 30 to 50 vol. % stillage.

The starch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids of the starch-containing material is convertedinto a soluble starch hydrolysate.

The process of the invention is conducted at a temperature below theinitial gelatinization temperature. Preferably the temperature at whichstep (a) is carried out is between 30-75° C., preferably between 45-60°C.

In a preferred embodiment step (a) and step (b) are carried out as asequential or simultaneous saccharification and fermentation process. Insuch preferred embodiment the process is typically carried at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In an embodiment simultaneous saccharification and fermentation iscarried out so that the sugar level, such as glucose level, is kept at alow level such as below 6 wt. %, preferably below about 3 wt. %,preferably below about 2 wt. %, more preferred below about 1 wt. %, evenmore preferred below about 0.5 wt. %, or even more preferred 0.25% wt.%, such as below about 0.1 wt. %. Such low levels of sugar can beaccomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich quantities of enzyme and fermenting organism to use. The employedquantities of enzyme and fermenting organism may also be selected tomaintain low concentrations of maltose in the fermentation broth. Forinstance, the maltose level may be kept below about 0.5 wt. % or belowabout 0.2 wt. %.

The process of the invention may be carried out at a pH in the rangebetween 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH4 to 5.

Starch-Containing Materials

Any suitable starch-containing starting material, including granularstarch, may be used according to the present invention. The startingmaterial is generally selected based on the desired fermentationproduct. Examples of starch-containing starting materials, suitable foruse in a process of present invention, include tubers, roots, stems,whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava,tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixturesthereof, or cereals, sugar-containing raw materials, such as molasses,fruit materials, sugar cane or sugar beet, potatoes, andcellulose-containing materials, such as wood or plant residues, ormixtures thereof. Contemplated are both waxy and non-waxy types of cornand barley.

The term “granular starch” means raw uncooked starch, i.e., starch inits natural form found in cereal, tubers or grains. Starch is formedwithin plant cells as tiny granules insoluble in water. When put in coldwater, the starch granules may absorb a small amount of the liquid andswell. At temperatures up to 50° C. to 75° C. the swelling may bereversible. However, with higher temperatures an irreversible swellingcalled “gelatinization” begins. Granular starch to be processed may be ahighly refined starch quality, preferably at least 90%, at least 95%, atleast 97% or at least 99.5% pure or it may be a more crude starchcontaining material comprising milled whole grain including non-starchfractions such as germ residues and fibers. The raw material, such aswhole grain, is milled in order to open up the structure and allowingfor further processing. Two milling processes are preferred according tothe invention: wet and dry milling. In dry milling whole kernels aremilled and used. Wet milling gives a good separation of germ and meal(starch granules and protein) and is often applied at locations wherethe starch hydrolysate is used in production of syrups. Both dry and wetmilling is well known in the art of starch processing and is equallycontemplated for the process of the invention.

The starch-containing material is reduced in particle size, preferablyby dry or wet milling, in order to expose more surface area. In anembodiment the particle size is between 0.05 to 3.0 mm, preferably0.1-0.5 mm, or so that at least 30%, preferably at least 50%, morepreferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.,ethanol, methanol, butanol); organic acids (e.g., citric acid, aceticacid, itaconic acid, lactic acid, gluconic acid); ketones (e.g.,acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂);antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B₁₂, beta-carotene); and hormones. In a preferredembodiment the fermentation product is ethanol, e.g., fuel ethanol;drinking ethanol, i.e., potable neutral spirits; or industrial ethanolor products used in the consumable alcohol industry (e.g., beer andwine), dairy industry (e.g., fermented dairy products), leather industryand tobacco industry. Preferred beer types comprise ales, stouts,porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer,low-alcohol beer, low-calorie beer or light beer. Preferred fermentationprocesses used include alcohol fermentation processes, as are well knownin the art. Preferred fermentation processes are anaerobic fermentationprocesses, as are well known in the art.

Fermenting Organisms

“Fermenting organism” refers to any organism, including bacterial andfungal organisms, suitable for use in a fermentation process and capableof producing desired a fermentation product. Especially suitablefermenting organisms are able to ferment, i.e., convert, sugars, such asglucose or maltose, directly or indirectly into the desired fermentationproduct. Examples of fermenting organisms include fungal organisms, suchas yeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae. Commercially available yeastinclude, e.g., Red Star™/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food Inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

Enzymes

Glucoamylase

The glucoamylase is preferably a glucoamylase of the invention. However,as mentioned above a glucoamylase of the invention may also be combinedwith other glucoamylases. The term “glucoamylase” (1,4-alpha-D-glucanglucohydrolase, EC 3.2.1.3) is an enzyme, which catalyzes the release ofD-glucose from the non-reducing ends of starch or related oligo- andpolysaccharide molecules.

The glucoamylase may added in an amount of 0.001 to 10 AGU/g DS,preferably from 0.01 to 5 AGU/g DS, such as around 0.1, 0.3, 0.5, 1 or 2AGU/g DS, especially 0.1 to 0.5 AGU/g DS or 0.02-20 AGU/g DS, preferably0.1-10 AGU/g DS.

Alpha-Amylase

The alpha-amylase may be of any origin. Preferred are alpha-amylases offungal or bacterial origin.

In a preferred embodiment the alpha-amylase is an acid alpha-amylase,e.g., fungal acid alpha-amylase or bacterial acid alpha-amylase. Theterm “acid alpha-amylase” means an alpha-amylase (EC 3.2.1.1) whichadded in an effective amount has activity optimum at a pH in the rangeof 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylases

A bacterial alpha-amylase may preferably be derived from the genusBacillus.

In a preferred embodiment the Bacillus alpha-amylase is derived from astrain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B.stearothermophilus, but may also be derived from other Bacillus sp.Specific examples of contemplated alpha-amylases include the Bacilluslicheniformis alpha-amylase (BLA) shown in SEQ ID NO: 4 in WO 99/19467,the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 5in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase (BSG)shown in SEQ ID NO: 3 in WO 99/19467. In an embodiment of the inventionthe alpha-amylase is an enzyme having a degree of identity of at least60%, preferably at least 70%, more preferred at least 80%, even morepreferred at least 90%, such as at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity to any of the sequences shownas SEQ ID NO: 1, 2, 3, 4, or 5, respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562,6,187,576, and 6,297,038 (hereby incorporated by reference) and includeBacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variantshaving a deletion of one or two amino acid in position 179 to 182,preferably a double deletion disclosed in WO 96/23873—see, e.g., page20, lines 1-10 (hereby incorporated by reference), preferablycorresponding to delta (181-182) compared to the wild-type BSGalpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed inWO 99/19467 or deletion of amino acids 179 and 180 using SEQ ID NO: 3 inWO 99/19467 for numbering (which reference is hereby incorporated byreference). Even more preferred are Bacillus alpha-amylases, especiallyBacillus stearothermophilus alpha-amylase, which have a double deletioncorresponding to delta (181-182) and further comprise a N193Fsubstitution (also denoted I181*+G182*+N193F) compared to the wild-typeBSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3disclosed in WO 99/19467.

The alpha-amylase may also be a maltogenic alpha-amylase. A “maltogenicalpha-amylase” (glucan 1,4-alpha-maltohydrolase, EC 3.2.1.133) is ableto hydrolyze amylose and amylopectin to maltose in thealpha-configuration. A maltogenic alpha-amylase from Bacillusstearothermophilus strain NCIB 11837 is commercially available fromNovozymes A/S, Denmark. The maltogenic alpha-amylase is described inU.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are herebyincorporated by reference.

Bacterial Hybrid Alpha-Amylases

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/19467), withone or more, especially all, of the following substitutions:G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacilluslicheniformis numbering). Also preferred are variants having one or moreof the following mutations (or corresponding mutations in other Bacillusalpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/ordeletion of two residues between positions 176 and 179, preferablydeletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO99/19467).

The bacterial alpha-amylase may be added in amounts as are well-known inthe art.

Fungal Alpha-Amylases

Fungal acid alpha-amylases include acid alpha-amylases derived from astrain of the genus Aspergillus, such as Aspergillus kawachii,Aspergillus niger, or Aspergillus oryzae alpha-amylases.

A preferred acid fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is preferably derived from a strain of Aspergillus oryzae. In thepresent disclosure, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e., more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in more detail in WO 89/01969 (Example 3). The acidAspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO2004/080923 (Novozymes) which is hereby incorporated by reference. Alsovariants of said acid fungal amylase having at least 70% identity, suchas at least 80% or even at least 90% identity, such as at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 1 in WO 2004/080923 are contemplated. A suitable commerciallyavailable acid fungal alpha-amylase derived from Aspergillus niger isSP288 (available from Novozymes A/S, Denmark).

In a preferred embodiment the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81:292-298, “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii”; and further as EMBL:#AB008370.

The fungal acid alpha-amylase may also be a wild-type enzyme comprisinga carbohydrate-binding module (CBM) and an alpha-amylase catalyticdomain (i.e., a none-hybrid), or a variant thereof. In an embodiment thewild-type acid alpha-amylase is derived from a strain of Aspergilluskawachii.

Fungal Hybrid Alpha-Amylases

In a preferred embodiment the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. ApplicationPublication no. 2005/0054071 (Novozymes) or U.S. application No.60/638,614 (Novozymes) which is hereby incorporated by reference. Ahybrid alpha-amylase may comprise an alpha-amylase catalytic domain (CD)and a carbohydrate-binding domain/module (CBM) and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in U.S. application No. 60/638,614 including Fungamyl variantwith catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 100 inU.S. application No. 60/638,614), Rhizomucor pusillus alpha-amylase withAthelia rolfsii AMG linker and SBD (SEQ ID NO: 101 in U.S. applicationNo. 60/638,614) and Meripilus giganteus alpha-amylase with Atheliarolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S. applicationNo. 60/638,614).

Other specific examples of contemplated hybrid alpha-amylases includethose disclosed in U.S. Application Publication no. 2005/0054071,including those disclosed in Table 3 on page 15, such as Aspergillusniger alpha-amylase with Aspergillus kawachii linker and starch bindingdomain.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™,LIQUOZYME™ X and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™L-40,000, DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ Ethyl, GC358,GC980, SPEZYME™ RSL, and SPEZYME™ DELTA AA (Genencor Int.), and the acidfungal alpha-amylase sold under the trade name SP288 (available fromNovozymes A/S, Denmark).

An acid alpha-amylase may according to the invention be added in anamount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS,especially 0.3 to 2 AFAU/g DS.

Production of Syrup

The present invention also provides a process of using a glucoamylase ofthe invention for producing syrup, such as glucose and the like, fromstarch-containing material. Suitable starting materials are exemplifiedin the “Starch-containing materials”-section above. Generally, theprocess comprises the steps of partially hydrolyzing starch-containingmaterial (liquefaction) in the presence of alpha-amylase and thenfurther saccharifying the release of glucose from the non-reducing endsof the starch or related oligo- and polysaccharide molecules in thepresence of glucoamylase of the invention.

Liquefaction and saccharification may be carried our as described abovefor fermentation product production.

The glucoamylase of the invention may also be used in immobilized form.This is suitable and often used for producing speciality syrups, such asmaltose syrups, and further for the raffinate stream of oligosaccharidesin connection with the production of fructose syrups, e.g., highfructose syrup (HFS).

Consequently, this aspect of the invention relates to a process ofproducing syrup from starch-containing material, comprising

(a) liquefying starch-containing material in the presence of analpha-amylase, and

(b) saccharifying the material obtained in step (a) using a glucoamylaseof the invention.

A syrup may be recovered from the saccharified material obtained in step(b).

Details on suitable conditions can be found above.

Brewing

A glucoamylase of the invention can also be used in a brewing process.The glucoamylase of the invention is added in effective amounts whichcan be easily determined by the skilled person in the art.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties. The present invention isfurther described by the following examples which should not beconstrued as limiting the scope of the invention.

Signal Peptide and Propeptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to19 of SEQ ID NO: 2 or 1 to 19 of SEQ ID NO: 4. The polynucleotides mayfurther comprise a gene encoding a protein, which is operably linked tothe signal peptide. The protein is preferably foreign to the signalpeptide and/or propeptide. In one aspect, the polynucleotide encodingthe signal peptide is nucleotides 1 to 57 of SEQ ID NO: 1 or 1 to 57 ofSEQ ID NO: 3.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examples.

EXAMPLES Glucoamylase Activity

Glucoamylase activity may be measured in AGI units or in GlucoamylaseUnits (AGU), or according to other suitable assays like e.g. acommercial assay kit from Kikkoman.

Glucoamylase Activity (AGI)

Glucoamylase (equivalent to amyloglucosidase) converts starch intoglucose. The amount of glucose is determined here by the glucose oxidasemethod for the activity determination. The method described in thesection 76-11 Starch—Glucoamylase Method with Subsequent Measurement ofGlucose with Glucose Oxidase in “Approved methods of the AmericanAssociation of Cereal Chemists”. Vol. 1-2 AACC, from AmericanAssociation of Cereal Chemists, (2000); ISBN: 1-891127-12-8.

One glucoamylase unit (AGI) is the quantity of enzyme which will form 1micro mole of glucose per minute under the standard conditions of themethod.

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch, concentration approx. 16 g dry matter/L.

Buffer: Acetate, approx. 0.04M, pH=4.3

pH: 4.3

Incubation temperature: 60° C.

Reaction time: 15 minutes

Termination of the reaction: NaOH to a concentration of approximately0.2 g/L (pH-9)

Enzyme concentration: 0.15-0.55 AAU/mL.

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as colorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine.

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nmGlucoamylase Activity Assay (Kikkoman)Product code: 60211This kit is for measurement of the glucose-forming activity in ricekoji.Assay Principle:

The substrate, 4-nitrophenyl-β-maltoside (G2-β-pNP) is degraded byglucoamylase or α-glucosidase into 4-nitrophenyl-β-glucoside(G1-(3-pNP). G1-6-pNP is further degraded into 4-nitrophenol (pNP) byβ-glucosidase in this kit. Reaction is performed at room temperature atpH about 4. The reaction is stopped by addition of sodium carbonate, andat the same time the solution becomes alkaline pH to maximize theabsorbance of pNP. The glucose-forming activity is measured byquantifying the pNP at 400 nm.

1) The measured response shows the G2-β-pNP degradation activity ofglucoamylase and α-glucosidase in the sample. This is thought to be theglucose forming activity in the sample.

2) The test can be used for rice koji extract without dialysis.

3) This assay is not affected by α-amylase in the sample.

Kit Components

Reagent Main component Amount substrate solution G2-β-pNP  60 ml enzymesolution β-glucosidase  60 ml stop solution sodium carbonate 120 ml

-   1) Mix “substrate solution” and “enzyme solution” of the kit at 1:1.-   2) Pipette 20 μl of the sample (or water as a blank) and transfer it    to a microtiter plate well. (duplicate)-   3) Add 60 μl of the substrate-enzyme mixture to the well.-   4) Incubate at room temperature for 20 min.-   5) Add 120 μl of the stop solution to the well.-   6) Read OD400 nm. # Net OD₄₀₀=OD₄₀₀(sample)−OD₄₀₀(blank)    1. Blank: Usually, the blank absorbance is less than 0.200.    2. Specificity: The response is not affected by glucose (up to 100    g/l) or α-amylase (725 U/ml).    3. Reproducibility: The CV of absorbance is less than 1% when the    same sample is analyzed 10 times.    4. Linear range: The net OD₄₀₀ up to 1.6 should be proportional to    the enzyme concentration.    5. Stability of color: The absorbance does not change for 2 h at 25°    C.    Wako Glucose Assay Kit (LabAssay Glucose, WAKO, Cat#298-65701).

LabAssay™ Glucose is a reagent kit for assay of glucose based on anenzymatic method with a combination of mutarotase and glucose oxidase.

α-D-glucose and β-D-glucose in solution maintain equilibrium in aconstant ratio. Glucose oxidase reacts only with β-D-glucose and doesnot react with α-D-glucose. Therefore, α-D-glucose is converted intoβ-D-glucose using mutarotase.

When a sample is mixed with the Chromogen Reagent, the alpha-form ofglucose in the sample is converted to beta-form by mutarotase.Beta-D-glucose is oxidized and yields hydrogen peroxide by glucoseoxidase (GOD). In the presence of peroxidase (POD), the formed hydrogenperoxide yields a red pigment by quantitative oxidation condensationwith phenol and 4-aminoantipyrine. The glucose concentration is obtainedby measuring absorbance of the red pigment.

The assay was performed in 96 wells microplates using a microplatereader with 505 nm wavelength filter. Assay was performed at 37° C. for5 min.

The principle of the assay method is further described in Miwa, et al.,1972, Clin. Chim. Acta., 37, 538-540.

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Chaetomium thermophilum (CBS144.50) was used as the source ofpolypeptides having glucoamylase activity. Aspergillus niger strainHowB112 was used for expression of the Chaetomium thermophilum genesencoding the polypeptides having glucoamylase activity. A. niger HowB112is an amdS (acetamidase) gene disrupted derivative of A. niger BO1.Strain HowB112 bears following genotypes: AMG⁻ (disrupted glucoamylasegene), ASA⁻ (disrupted acid stable amylase gene) and tgs⁻ (disruptedalpha-1,6-transglucosidase gene).

Media and Solutions

YMD medium was composed of 0.3% yeast extract, 0.5% peptone, of 0.3%malt extract and 5% maltodextrin.

PDA agar plates were composed of potato infusion (potato infusion wasmade by boiling 300 g of sliced (washed but unpeeled) potatoes in waterfor 30 minutes and then decanting or straining the broth throughcheesecloth. Distilled water was then added until the total volume ofthe suspension was one liter, followed by 20 g of dextrose and 20 g ofagar powder. The medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998).

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionized water to 1 liter.

YPG medium contained 0.4% of yeast extract, 0.1% of KH2PO4, 0.05% ofMgSO4.7H₂O, 1.5% glucose in deionized water

COVE-N-gly slants were composed of 218 g sorbitol, 10 g glycerol, 2.02 gKNO3, 50 ml COVE salt solution, 25 g agar powder and deionized water to1 liter.

COVE plates for protoplast regeneration were composed of 342 g ofsucrose, 20 g of agar powder, 20 ml of COVE salt solution, and deionizedwater to 1 liter. The medium was sterilized by autoclaving at 15 psi for15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998). The medium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCl,were added.

COVE top agarose were composed of 342.3 g sucrose, 20 ml COVE saltsolution, 6 g GTG agarose (SeaKem, Cat#50070) and deionized water to 1liter. The medium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C., and 10 mM acetamide and 15 mM CsCl wereadded.

COVE-2 plate for isolation were composed of 30 g sucrose, 20 ml COVEsalt solution, 30 g agar powder and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide was added.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 ml of COVE trace metal solution, and deionized water to 1liter.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Example 1 Chaetomium thermophilum Genomic DNA Extraction

Chaetomium thermophilum strain CBS144.50 was inoculated onto a PDA plateand incubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen in liquid nitrogen.Frozen mycelia were ground, by a mortar and a pestle, to a fine powder,and genomic DNA was isolated using DNeasy® Plant Maxi Kit (QIAGEN Inc.,Valencia, Calif., USA) following the manufacturer's instruction.

Example 2 Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using ILLUMINA®GA2 System (Illumina, Inc., San Diego, Calif., USA). The raw reads wereassembled at BGI using in house program SOAPdenovo. The assembledsequences were analyzed using standard bioinformatics methods for genefinding and functional prediction. Briefly, gene ID (Parra et al., 2000,Genome Research 10(4):511-515) was used for gene prediction. Blastallversion 2.2.10 (National Center for Biotechnology Information (NCBI)Bethesda, Md., USA) and HMMER version 2.1.1 (National Center forBiotechnology Information (NCBI) Bethesda, Md., USA) were used topredict function based on structural homology. The family GH15glucoamylase candidates were identified directly by analysis of theBlast results. A gene (Munch and Krogh, 2006, BMC Bioinformatics 7:263)and SignalP (Nielsen et al., 1997, Protein Engineering 10:1-6) were usedto identify starting codons. SignalP was further used to estimate lengthof signal peptide. Pepstats (European Bioinformatics Institute, Hinxton,Cambridge CB10 1SD, UK) was used to estimate isoelectric point ofproteins, and molecular weight.

2 annotated GH15 glucoamylase genes (shown in Table 1) were selected forexpression cloning.

TABLE 1 GH15 glucoamylase genes from Chaetomium thermophilum Gene nameDNA sequence AMG56053-1 SEQ ID: 1 AMG56053-2 SEQ ID: 3

Example 3 Cloning of 2 GH15 Glucoamylase Genes from the Chaetomiumthermophilum Genomic DNA

Based on the DNA information obtained from genome sequencing,oligonucleotide primers, shown below in table 2, were designed toamplify the 2 GH15 glucoamylase genes (SEQ ID: 1 and 3) from the genomicDNA of Chaetomium thermophilum CBS144.50. Primers were synthesized byInvitrogen (Invitrogen, Beijing, China).

TABLE 2 Primers to amplify full-length glucoamylase genes from Chaetomium thermophilum genomic DNA Related  SEQ ID Primer nameSequence (5′-3′) SEQ ID  AMG56053- acacaactggggaTCACC  NO: 5 1_C505_bamATGCCTCTCTCGTCACTC SEQ ID  AMG56053- ccctctagatctcgaGAAATCTAA NO: 61_C505_xho CGCCACGAAGCCTCCT SEQ ID  AMG56053- acacaactggggatccaccATGCCNO: 7 2_P355_BamH ATTGATTAAAACACTGGCATTAAC SEQ ID  AMG56053-gtcaccctctagatctcgagACTC NO: 8 2_P355_BgIII GGCCAATCTCGGCTTTA

Upper characters represent the 5′- and 3′-regions of the to be amplifiedgenes, while lower cases were homologous to the vector sequences atinsertion sites of pCaHj505 vector. The expression vector pCaHj505containins the TAKA-amylase promoter derived from Aspergillus oryzae andthe Aspergillus niger glucoamylase terminator elements. FurthermorepCaHj505 has pUC18 derived sequences for selection and propagation in E.coli, and an amdS gene, which encodes an acetoamidase gene derived fromAspergillus nidulans for selection of an amds⁺ Aspergillus transformant.

For each gene, 20 pmol of primer pair (each of the forward and reverse)were used in a PCR reaction composed of 2 μl of Chaetomium thermophilumCBS144.50 genomic DNA, 10 μl of 5×GC Buffer, 1.5 μl of DMSO, 2.5 mM eachof dATP, dTTP, dGTP, and dCTP, and 0.6 unit of Phusion™ High-FidelityDNA Polymerase (FinnzymesOy, Espoo, Finland) in a final volume of 50 μl.The amplification was performed using a Peltier Thermal Cycler (M JResearch Inc., South San Francisco, Calif., USA) programmed fordenaturing at 98° C. for 1 minutes; 10 cycles of denaturing at 98° C.for 15 seconds, annealing at 65° C. for 30 seconds, with 1° C. decreaseper cycle and elongation at 72° C. for 90 seconds; and another 26 cycleseach at 98° C. for 15 seconds, 60° C. for 30 seconds and 72° C. for 90seconds; final extension at 72° C. for 10 minutes. The heat block thenwent to a 4° C. soak cycle.

The PCR products were isolated by 0.7% agarose gel electrophoresis using90 mM Tris-borate and 1 mM EDTA (TBE) buffer where product bands atexpected size of each PCR reaction were visualized under UV light. ThePCR products were then purified from solution by using a GFX PCR DNA andGel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) accordingto the manufacturer's instructions.

TABLE 3 Size of PCR products in Example 3 Gene name Size of PCR productAMG56053-1 2 kb AMG56053-2 2 kb

Plasmid pCaHj505 was digested with BamHI and XhoI, isolated by 0.7%agarose gel electrophoresis using TBE buffer, and purified using anIllustra™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

An In-Fusion™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pCaHj505.

The PCR products and the digested vector were ligated together using anIN-FUSION™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) resulting in plasmids in Table 4respectively, in which transcription of Humicola insolens GH15glucoamylase genes was under the control of a TAKA-amylase promoter fromAspergillus otyzae. The cloning operation was according to themanufacturer's instruction. In brief, for each ligation reaction 30 ngof with BamHI and XhoI digested pCaHj505 and 60 ng of purified PCRproducts were added to the reaction vial and resuspended with the powderin a final volume of 10 μl with addition of deionized water. Thereactions were incubated at 37° C. for 15 minutes and then 50° C. for 15minutes. 3 μl of the reaction were transformed into E. coli TOP10competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China)according to the manufacturer's protocol and plated onto LB platessupplemented with 0.1 mg of ampicillin per ml. After incubating at 37°C. overnight, colonies were seen growing on the LB ampicillin plates. E.coli transformants containing expression constructs were detected bycolony PCR and confirmed by DNA sequencing with vector primers (bySinoGenoMax Company Limited, Beijing, China). Plasmid DNApAMG56053-1_C505 and pAMG56053-2_C505 for expression in A. niger wereextracted from correct E. coli transformants, by using a QIAprep SpinMiniprep Kit (QIAGEN Inc., Valencia, Calif., USA).

TABLE 4 Plasmid (Expression constructs) in Example 3 Gene name PlasmidAMG56053-1 pAMG56053-1_C505 AMG56053-2 pAMG56053-2_C505

Example 4 Expression of Chaetomium thermophilum GH15 Glucoamylse Genesin Aspergillus niger

An agar slant (COVE-N-gly) was inoculated with spores of Aspergillusniger HowB112, and grown at 32° C. until it was completely sporulated.The spores were resuspended in 5-10 ml of sterile 0.05% tween20 water.About 10⁸ spores were transferred to a 500 ml baffled shake flaskcontaining 100 ml YPG medium with 10 mM NaNO₃, and incubated at 32° C.for 16 hrs at 99 rpm in Innova shaker. Then the mycelia were harvestedfor protoplasts preparation. Aspergillus niger HowB112 protoplastspreparation and transformation were done according to the methoddescribed in patent WO2004/111218 or EP 238023. 10 μg ofpAMG56053-1_C505 was used to transform Aspergillus niger HowB112separately.

The Aspergillus niger HowB112 transformants with pAMG56053-1_C505 wereselected on the COVE plates for protoplast regeneration (described inthe Media and Solution part). About 10 transformants were observed onthe selective plates for each transformation. Six transformants fromeach transformation were isolated on COVE-2 plate for 3-4 days at 32° C.

After isolation those six transformants for each transformation wereinoculated separately into 3 ml of YMD medium in 24-well plate andincubated at 30° C., 220 rpm. After 3 days incubation, 20 μl ofsupernatant from each culture were analyzed on NuPAGENovex 4-12%Bis-Tris Gel w/MES (Invitrogen Corporation, Carlsbad, Calif., USA)according to the manufacturer's instructions. The resulting gel wasstained with Instant Blue (Expedeon Ltd., Babraham Cambridge, UK).SDS-PAGE profiles of the cultures showed that they had the exceptedprotein bands of expression products ofpAMG56053-1_C505. The expressionproduct number and expression strain number was shown in Table 5.

TABLE 5 Expression strains Expression construct Expression productExpression strain pAMG56053-1_C505 P23S9D (SEQ ID NO: 2) O4J6F

Example 5 Fermentation of A. niger Expression Strains

A slant of O4J6F was washed with 10 ml of YMD and inoculated into a 2liter flask containing 400 ml of YMD medium to generate broth forcharacterization of the enzyme. The culture was incubated at 30° C. onshaker at 150 rpm. The culture was harvested on day 3 and filtered usinga 0.45 μm DURAPORE Membrane (Millipore, Bedford, Mass., USA). Thefiltered culture broth was used for enzyme characterization.

Example 6 Characterization of Glucoamylases

Substrate: 1% soluble starch (Sigma S-9765) in deionized water

Reaction buffer: 0.1M Acetate buffer at pH4.3

Glucose concentration determination kit: Wako glucose assay kit(LabAssay glucose, WAKO, Cat#298-65701).

Reaction Condition:

20 μl soluble starch and 50 μl acetate buffer at pH5.3 were mixed. 30 μlenzyme solution (50 μg enzyme protein/mil) was added to a final volumeof 100 μl followed by incubation at 37° C. for 15 min.

The glucose concentration is determined by Wako kits.

All the experiments were carried out in parallel.

Temperature Optimum.

To assess the temperature profile, the Reaction condition assaydescribed above was performed at 20, 30, 40, 50, 60, 70, 80, and 90° C.The results are shown in table 6.

TABLE 6 Temperature optimum Temperature (° C.) 20 30 40 50 60 70 80 90Relative P23S9D 46.2 52.0 63.6 82.0 91.5 100 91.7 73.0 activity (%)From the result it can be seen that the optimal temperature for P23S9Datthe given conditions is around 70° C.Heat Stability:

To assess the heat stability of these glucoamylases the Reactioncondition assay was modified in that the enzyme solution and acetatebuffer was preincubated at 70° C. for 0, 10, 30, 60, and 120 minutes.Following the incubation 20 μl of starch was added to the solution andthe assay was performed as described above.

The results were shown in table 7.

TABLE 7 Heat stability Incubation time (min) 0 10 30 60 120 RelativeP23S9D (70° C.) 100 42.1 41.2 43.6 42.5 activity (%)pH Optimum:

To assess the pH optimum of the glucoamylases the Reaction conditionassay described above was performed at pH 2.0, 3.0, 4.0, 5.0, 6.0 7.0,8.0, 9.0, 10.0 and 11.0. Instead of using the acetate buffer describedin the Reaction condition assay the following buffer was used 100 mMSuccinic acid, HEPES, CHES, CAPSO, 1 mM CaCl₂, 150 mM KCl, 0.01% TritonX-100, pH adjusted to 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0,10.0 or 11.0 with HCl or NaOH. The results were shown in table 8.

TABLE 8 pH optimum pH 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 RelativeP23S9D 58.5 53.6 86.8 99.5 97.7 98.6 100 68.8 69.7 59.3 activity (%)

From the result it can be seen that P23S9D has a very broad pH profile.

pH Stability:

30 μl enzyme solution (50 μg/ml) and 50 μl buffer at was mixed andpreincubated for 0, 10, 30, 60, 120 mins. After preincubation, 20 μlsoluble starch in a final volume of 100 μl was incubated at 37° C. for15 min.

Finally the glucose concentration was determined using the Wako kits.The results are shown in table 9.

TABLE 9 pH stability Incubation time (min) 0 10 30 60 120 RelativeP23S9D (pH 5) 100 99.4 99.2 98.6 98.6 activity (%)From the result it can be seen that the glucoamylase is stable at acidiccondition under the conditions tested.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The present invention is further described by the following numberedparagraphs.

[1] An isolated polypeptide having glucoamylase activity, selected fromthe group consisting of:

-   -   (a) a polypeptide having at least 76% sequence identity to the        mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;    -   (b) a polypeptide encoded by a polynucleotide that hybridizes        under medium stringency conditions, high stringency conditions,        or very high stringency conditions with (i) the mature        polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:        3, (ii) the cDNA sequence thereof, or (iii) the full-length        complement of (i) or (ii);    -   (c) a polypeptide encoded by a polynucleotide having at least        76% sequence identity to the mature polypeptide coding sequence        of SEQ ID NO: 1 or SEQ ID NO: 3, or the cDNA sequence thereof;    -   (d) a variant of the mature polypeptide of SEQ ID NO: 2 or SEQ        ID NO: 4 comprising a substitution, deletion, and/or insertion        at one or more positions; and    -   (e) a fragment of the polypeptides of (a), (b), (c), or (d) that        has glucoamylase activity.        [2] The polypeptide of paragraph 1, having at least 78%, at        least 80%, at least 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 96%, at        least 97%, at least 98%, at least 99%, or 100% sequence identity        to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.        [3] The polypeptide of paragraph 1 or 2, which is encoded by a        polynucleotide that hybridizes under medium-high stringency        conditions, high stringency conditions, or very high stringency        conditions with (i) the mature polypeptide coding sequence of        SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence thereof,        or (iii) the full-length complement of (i) or (ii).        [4] The polypeptide of any of paragraphs 1-3, which is encoded        by a polynucleotide having at least 78%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%, or 100% sequence identity to the mature        polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or        the cDNA sequence thereof.        [5] The polypeptide of any of paragraphs 1-4, comprising or        consisting of SEQ ID NO: 2 or SEQ ID NO: 4, or the mature        polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.        [6] The polypeptide of paragraph 5, wherein the mature        polypeptide is amino acids 20 to 634 of SEQ ID NO: 2 or amino        acids 20 to 631 of SEQ ID NO: 4.        [7] The polypeptide of any of paragraphs 1-4, which is a variant        of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4        comprising a substitution, deletion, and/or insertion at one or        more (several) positions.        [8] The polypeptide of paragraph 1, which is a fragment of SEQ        ID NO: 2 or SEQ ID NO: 4, wherein the fragment has glucoamylase        activity activity.        [9] An isolated polypeptide comprising a catalytic domain        selected from the group consisting of:    -   (a) a catalytic domain having at least 76% sequence identity to        amino acids 33 to 505 of SEQ ID NO: 2 or amino acids 28 to 504        of SEQ ID NO: 4;    -   (b) a catalytic domain encoded by a polynucleotide that        hybridizes under medium stringency conditions with (i)        nucleotides 97 to 1609 of SEQ ID NO: 1 or nucleotides 82 to 1680        of SEQ ID NO: 3, (ii) the cDNA sequences thereof, or (iii) the        full-length complement of (i) or (ii);    -   (c) a catalytic domain encoded by a polynucleotide having at        least 76% sequence identity to the catalytic domain of SEQ ID        NO: 1 or of SEQ ID NO: 3 or the cDNA sequences thereof;    -   (d) a variant of amino acids 33 to 505 of SEQ ID NO: 2 or amino        acids 28 to 504 of SEQ ID NO: 4 comprising a substitution,        deletion, and/or insertion at one or more (e.g., several)        positions; and    -   (e) a fragment of the catalytic domain of (a), (b), (c), or (d)        that has glucoamylase activity.        [10] The polypeptide of paragraph 9, further comprising a        carbohydrate binding domain.        [11] A composition comprising the polypeptide of any of        paragraphs 1-10.        [12] A composition according to paragraph 11, comprising an        alpha-amylase and a polypeptide of any of paragraphs 1-10.        [13] A use of a polypeptide of any of paragraphs 1-10 for        production of syrup and/or a fermentation product.        [14] The use according to paragraph 13, wherein the starting        material is gelatinized or un-gelatinized starch-containing        material.        [15] A use of a polypeptide of any of paragraphs 1-10 for        brewing.        [16] A process of producing a fermentation product from        starch-containing material comprising the steps of:    -   (a) liquefying starch-containing material;    -   (b) saccharifying the liquefied material; and    -   (c) fermenting with a fermenting organism;        wherein step (b) is carried out using at least a glucoamylase of        any of paragraphs 1-10.        [17] A process of producing a fermentation product from        starch-containing material, comprising the steps of:    -   (a) saccharifying starch-containing material at a temperature        below the initial gelatinization temperature of said        starch-containing material; and    -   (b) fermenting with a fermenting organism,        wherein step (a) is carried out using at least a glucoamylase of        any of paragraphs 1-10.        [18] An isolated polynucleotide encoding the polypeptide of any        of paragraphs 1-10.        [19] A nucleic acid construct or expression vector comprising        the polynucleotide of paragraph 18 operably linked to one or        more control sequences that direct the production of the        polypeptide in an expression host.        [20] A recombinant host cell comprising the polynucleotide of        paragraph 18 operably linked to one or more control sequences        that direct the production of the polypeptide.        [21] A method of producing the polypeptide of any of paragraphs        1-10, comprising:    -   (a) cultivating a cell, which in its wild-type form produces the        polypeptide, under conditions conducive for production of the        polypeptide; and    -   (b) recovering the polypeptide.        [22] A method of producing a polypeptide of any of paragraphs        1-10, comprising:    -   (a) cultivating the host cell of paragraph 20 under conditions        conducive for production of the polypeptide; and    -   (b) recovering the polypeptide.        [23] A transgenic plant, plant part or plant cell comprising a        polynucleotide encoding the polypeptide of any of paragraphs        1-10.        [24] A method of producing a polypeptide of any of paragraphs        1-10, comprising:    -   (a) cultivating the transgenic plant or plant cell of paragraph        23 under conditions conducive for production of the polypeptide;        and    -   (b) recovering the polypeptide.

The invention claimed is:
 1. A process of producing a fermentationproduct from starch-containing material comprising the steps of: (a)liquefying starch-containing material; (b) saccharifying the liquefiedmaterial; and (c) fermenting with a fermenting organism; wherein step(b) is carried out by contacting the liquefied material with at least aglucoamylase having at least 85% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, and wherein thefermentation product is ethanol.
 2. The process of claim 1 wherein theglucoamylase has at least 90% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 3. The process of claim 1 wherein theglucoamylase has at least 95% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 4. The process of claim 1 wherein theglucoamylase has at least 97% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 5. The process of claim 1 wherein theglucoamylase has at least 98% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 6. The process of claim 1 wherein theglucoamylase has at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 7. The process of claim 1 wherein theglucoamylase has at least 90% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 8. The process of claim 1 wherein theglucoamylase has at least 95% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 9. The process of claim 1 wherein theglucoamylase has at least 97% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 10. The process of claim 1 wherein theglucoamylase has at least 98% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 11. The process of claim 1 wherein theglucoamylase has at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 12. The process of claim 1, wherein thefermenting organism is yeast.
 13. A process of producing a fermentationproduct from starch-containing material comprising the steps of: (a)saccharifying starch-containing material at a temperature below theinitial gelatinization temperature of said starch-containing material;and (c) fermenting with a fermenting organism; wherein step (a) iscarried out by contacting the starch-containing material with at least aglucoamylase having at least 85% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, and wherein thefermentation product is ethanol.
 14. The process of claim 13 wherein thepolypeptide has at least 90% sequence identity to the mature polypeptideof SEQ ID NO:
 2. 15. The process of claim 13 wherein the polypeptide hasat least 95% sequence identity to the mature polypeptide of SEQ ID NO:2.
 16. The process of claim 13 wherein the polypeptide has at least 97%sequence identity to the mature polypeptide of SEQ ID NO:
 2. 17. Theprocess of claim 13 wherein the polypeptide has at least 98% sequenceidentity to the mature polypeptide of SEQ ID NO:
 2. 18. The process ofclaim 13 wherein the polypeptide has at least 99% sequence identity tothe mature polypeptide of SEQ ID NO:
 2. 19. The process of claim 13wherein the polypeptide has at least 90% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 20. The process of claim 13 wherein thepolypeptide has at least 95% sequence identity to the mature polypeptideof SEQ ID NO:
 4. 21. The process of claim 13 wherein the polypeptide hasat least 97% sequence identity to the mature polypeptide of SEQ ID NO:4.
 22. The process of claim 13 wherein the polypeptide has at least 98%sequence identity to the mature polypeptide of SEQ ID NO:
 4. 23. Theprocess of claim 13 wherein the polypeptide has at least 99% sequenceidentity to the mature polypeptide of SEQ ID NO:
 4. 24. The process ofclaim 13, wherein the fermenting organism is yeast.